A semi-automated microscopic image analysis method for scoring Ki-67 nuclear immunostaining.

Nuclear proliferation marker MIB-1 (Ki-67) immunohistochemistry (IHC) is used to examine tumor cell proliferation. However, the diagnostic or prognostic value of the Ki-67 nuclear staining intensity and location, defined as nuclear gradient (NG), has not been assessed. This study examined the potential association between Ki-67 NG and cell cycle phases and its effect on the prognosis of pulmonary typical carcinoid (PTC) tumors. We propose a method for classifying the NG of Ki-67 during the cell cycle and compare the results between PTC, pulmonary adenocarcinoma (PAD), and breast ductal carcinoma (BDC). A literature review and objective analysis of IHC-stained paraffin sections were used to determine the Ki-67 labeling index and composed a stratification of the NG into NG1, NG2, and NG3/4 categories. A semi-automated image analysis protocol was established to determine the Ki-67 NG in PTC, PAD, and BDC. High intraobserver consistency and moderate interobserver agreement were achieved in the determination of Ki-67 NG in tumor specimens. NG1 and NG2 were lower in PTC than in PAD and BDC. Cox multivariate analysis of PTC after adjusting for age and number of metastatic lymph nodes showed that Ki-67 NG1 and NG2 significantly predicted clinical outcomes. The semi-automated method for quantification of Ki-67 nuclear immunostaining proposed in this study could become a valuable diagnostic and prognostic tool in PTC.

A semi-automated microscopic image analysis method for scoring Ki-67 nuclear immunostaining

Nuclear proliferation marker MIB-1 (Ki-67) immunohistochemistry (IHC) is used to examine tumor cell proliferation. However, the diagnostic or prognostic value of the Ki-67 nuclear staining intensity and location, defined as nuclear gradient (NG), has not been assessed. This study examined the potential association between Ki-67 NG and cell cycle phases and its effect on the prognosis of pulmonary typical carcinoid (PTC) tumors. We propose a method for classifying the NG of Ki-67 during the cell cycle and compare the results between PTC, pulmonary adenocarcinoma (PAD), and breast ductal carcinoma (BDC). A literature review and objective analysis of IHC-stained paraffin sections were used to determine the Ki-67 labeling index and composed a stratification of the NG into NG1, NG2, and NG3/4 categories. A semi-automated image analysis protocol was established to determine the Ki-67 NG in PTC, PAD, and BDC. High intraobserver consistency and moderate interobserver agreement were achieved in the determination of Ki-67 NG in tumor specimens. NG1 and NG2 were lower in PTC than in PAD and BDC. Cox multivariate analysis of PTC after adjusting for age and number of metastatic lymph nodes showed that Ki-67 NG1 and NG2 significantly predicted clinical outcomes. The semi-automated method for quantification of Ki-67 nuclear immunostaining proposed in this study could become a valuable diagnostic and prognostic tool in PTC.

Physicochemical, pharmacokinetic, and pharmacodynamic characterization of isradipine tablets for controlled release.

Isradipine is a dihydropyridine calcium channel blocker (CCB) commonly used as vasodilator with antihypertensive properties. A remote-controlled release formulation for isradipine would substantially improve the clinical outcomes of the patients requiring chronic long-term treatment. In this work, sustained release (SR) tablets of isradipine, composed of hydroxypropylmethyl cellulose (HPMC), have been produced by wet granulation and their in vitro and in vivo characterization was compared to a conventional tablet dosage form of immediate release (IR) as preliminary assessment. Tablets composed of 15.0% (wt/wt) HPMC exhibited a SR profile over a period of 24 hours. The release of isradipine followed a Fickian diffusion pattern obeying to the first order kinetics and the extent of absorption was even higher in comparison to the developed conventional tablets, which showed immediate drug release. In vivo studies were carried out in rabbits, showing that the extent of isradipine absorption from the developed tablets was higher in comparison to IR tablets due to the modified release profile obtained for the former (p < 0.05). Our results suggest that SR tablets of isradipine are an efficient solid dosage form to overcome the limitations encountered in conventional IR tablets.

Distribution pattern(s) of sperm protein at 22 kDa (SP22) on fresh, cooled and frozen/thawed equine spermatozoa and expression of SP22 in tissues from the testes and epididymides of normal stallions.

The objectives of this study were to (i) verify localization of SP22 on fresh, cooled, and frozen/thawed equine spermatozoa and to (ii) determine SP22 mRNA and protein expression in equine testicular and epididymal tissues. Immunocytochemistry and Western blots were performed on the spermatozoa samples. Northern blots and Western blots were performed on the tissue samples. The immunocytochemistry revealed the presence of SP22 in all samples tested. The fresh spermatozoa stained predominantly over the equatorial segment as did the samples cooled for 1 and 2 days. The samples cooled for 3 days, and the frozen/thawed samples had an increased proportion of no staining. The Western blots revealed SP22 was present on all semen samples tested. The Northern blot of the tissues revealed a 1.0 kb mRNA transcript present in each of the tissues, and the Western blot revealed the presence of SP22 in each of the tissues. As expected, SP22 was found to be altered on cooled and frozen/thawed spermatozoa. Our results suggest that the equatorial pattern is the normal pattern in spermatozoa, while a complete loss of SP22 from the surface of spermatozoa seems to be the staining pattern indicating the most extreme abnormality with scattered staining of the head indicating intermediate damage.

Morphological and functional preservation of pre-antral follicles after vitrification of macaque ovarian tissue in a closed system.

STUDY QUESTION: What are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles? SUMMARY ANSWER: The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system. WHAT IS KNOWN ALREADY: For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers. STUDY DESIGN, SIZE, DURATION: Non-human primate model, n = 4, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (48 h) and isolated secondary follicle culture (6 weeks). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm(3)) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/4, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a 40°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed for histological analysis and cultured with bromodeoxyuridine (BrdU). Secondary follicles from VEG tissues were encapsulated and cultured (n = 24/treatment/animal). Follicle health, diameter and steroid [progesterone, androstenedione (A4), estradiol (E2)] production were analyzed weekly. MAIN RESULTS AND THE ROLE OF CHANCE: Dense stroma and intact pre-antral follicles were observed using VS containing 27% glycerol, 27% EG and 0.8% polymers with cooling in LN2 vapor and a two-step warming. Higher cooling and warming rates led to fracturing. BrdU uptake was evident in granulosa cells of growing follicles in fresh and vitrified tissues. Secondary follicles from fresh tissues (70 ± 12%) and tissues vitrified with VEG (52 ± 2%) showed similar survival rates (all data: mean ± SEM; P > 0.05). For both groups, the initial follicle diameter was similar and increased (P < 0.05) by Week 3, but diameters in vitrified follicles were smaller (P < 0.05) by Week 6 (566 ± 27 µm) than those of the fresh follicles (757 ± 26 µm). Antrum formation rates were lower (P < 0.05) for vitrified (37 ± 6%) relative to fresh (64 ± 8%) follicles. There was no significant change in levels in culture media of E2, P4 and A4 between fresh and VEG groups at any time point during culture. LIMITATIONS, REASONS FOR CAUTION: Only in vitro studies are reported. Future in vivo tissue transplantation studies will be needed to confirm long-term function and fertility potential of vitrified ovarian tissues. WIDER IMPLICATIONS OF THE FINDINGS: This is the first demonstration of antral follicle development during 3D culture following ovarian tissue vitrification in a closed system using primate ovarian tissue. While diminished antrum formation and slower growth in vitro reflect residual cryodamage, continued development of ovarian tissue vitrification based on cryobiology principles using a non-human primate model will identify safe, practical and efficient protocols for eventual clinical use. Tissue function following heterotopic transplantation is currently being examined. STUDY FUNDING/COMPETING INTEREST(S): National Institutes of Health (NIH) Oncofertility Consortium UL1 RR024926 (1RL1-HD058293, HD058295, PL1 EB008542), the Eunice Kennedy Shriver NICHD/NIH (U54 HD018185) and ONPRC 8P51OD011092-53. G.M.F. works for the company that makes the polymers used in the current study.

Preventing Microbial Contamination during Long-Term In Vitro Culture of Human Granulosa-Lutein Cells: An Ultrastructural Analysis.

Purpose. To investigate whether the addition of antibiotic/antimycotic during human granulosa-lutein cells (GLCs) isolation and cell-plating procedures prevents microbial contamination after 144 h of culture and also evaluate the effects of contamination on GLCs ultrastructure and steroid secretion. Methods. GLCs obtained from five women submitted to assisted reproductive techniques (ARTs) were isolated with PBS supplemented with antibiotic/antimycotic or PBS nonsupplemented and cultured for 144 h. GLCs were evaluated by transmission electron microscopy (TEM), and estradiol (E2) and progesterone (P4) secretion was assayed by chemiluminescence. Results. Although no contaminating microorganisms were identified by light microscopy, TEM analyses revealed several bacterial colonies in culture dishes of GLCs isolated with only PBS. Bacterial contamination disrupted the adherence of the GLCs to the culture plate interfering with monolayer formation affecting the growth pattern of GLCs. Various cellular debris and bacteria were observed, and no organelles were found in the cytoplasm of infected cells. While bacterial contamination decreased estradiol media levels, it increased progesterone, as compared with noncontaminated group. Conclusion. Taken together, our data showed that the addition of a high dose of antibiotic/antimycotic during the isolation and cell-plating procedures prevents microbial contamination of long-term GLCs culture as its effects on cells growth and function in vitro.

The karyotype of Holoaden luederwaldti (Anura, Strabomantidae), with report of natural triploidy.

The genus Holoaden includes three species described so far, but the only published cytogenetic data is from Holoaden bradei, with the karyotype 2n = 18, based on conventional staining. In the present paper we report, for the first time, data on chromosomes of H. luederwaldti, which presented 2n = 18 and a case of natural triploidy, with 2n = 3x = 27. In this sample, another karyotypic variation was observed due to the occurrence of two types of chromosome 8, which present submetacentric or subtelocentric morphologies. Homomorphic subtelocentric or heteromorphic condition was observed among the diploid specimens, whereas the triploid had one submetacentric and two subtelocentric chromosomes 8. In all specimens, Ag-NOR was located in the long arms of chromosomes 8, at the interstitial region when subtelocentric, or in the proximal region when submetacentric, confirmed by fluorescent in situ hybridization with the HM123 probe. The C bands showed centromeric distribution and distribution at Ag-NOR site. The centromeric heterochromatin was fluorescent with DAPI staining, whereas the Ag- NOR displayed bright fluorescence with CMA3. Fluorescent in situ hybridization using a telomeric probe labelled exclusively the telomere regions. Although the same 2n = 18 chromosome numbers have been observed in H. luederwaldti and H. bradei, some differences in both karyotypes can be visualized, mainly with regard to the morphology of the last chromosome pairs.

The use of microsensors to study the role of the loading rate and surface velocity on the growth and the composition of nitrifying biofilms.

The good composition and activity of biofilms are very important for successful operation and control of fixed-film biological reactors employed in liquid effluents treatment. During the last decade, microsensors have been applied to study microbial ecology. These sensors could provide information regarding the microbial activity concerning nitrification and denitrification that occur inside biofilms. Other techniques of molecular biology, such as fluorescence in situ hybridization (FISH), have also contributed to this matter because their application aids in the identification of the bacterial populations that compose the biofilms. The focus of this paper was to study the loading rate and surface velocity to promote the development of nitrifying biofilms in three distinct flow cells that were employed in the post treatment of a synthetic wastewater simulating the effluent from a UASB (Upflow Anaerobic Sludge Blanket) reactor. Using the FISH technique, it was found that the population of ammonia-oxidizing-bacteria was greater than that of nitrite-oxidizing-bacteria; this was also supported by the lower production of nitrate determined by physicochemical and microsensor analyses. It was verified that the loading rate and surface velocity that promoted the greatest nitrogen removal were 0.25 g N-amon m(-2)biofilm day(-1) and 1 m h(-1), respectively.

Karyotypic similarity among Barycholos ternetzi and five species of the genus Eleutherodactylus from southeastern Brazil (Anura, Brachycephalidae).

Comparative cytogenetic analyses were carried out in six species of Brachycephalidae from southeastern Brazil. Barycholos ternetzi, Eleutherodactylus binotatus, Eleutherodactylus guentheri, Eleutherodactylus juipoca, Eleutherodactylus parvus and Eleutherodactylus sp. have 2n=22 karyotypes with a marked variation in the morphology of chromosome pairs 8, 10 and 11, which are of telocentric or metacentric types, resulting in FN=38, 40 and 44. Eleutherodactylus have a single chromosome pair bearing Ag-NOR, i.e. pair 1 in E. binotatus, pair 6 in E. guentheri and E. parvus, and pair 11 in E. juipoca and Eleutherodactylus sp. In contrast, B. ternetzi showed Ag-positive sites in the chromosome pairs 1, 4, 5, 9 and 11, and only one to three labelings per metaphase in each individual. Nevertheless, the main chromosome pair with Ag-NOR in the species seems to be the 11th, like in E. juipoca and Eleutherodactylus sp. The NOR site was confirmed by fluorescence in situ hybridization (FISH) technique in E. binotatus and in B. ternetzi, bearing 1p1p and 9p11p11p Ag-NOR pattern, respectively. All the species exhibited predominantly centromeric C-banding pattern, but interstitial bands have also been observed in some cases. In E. binotatus, there is an indication of geographical difference in the distribution of the interstitial C-bands. The fluorochromes GC-specific chromomycin A(3) (CMA(3)) and AT-specific 4',6-diamidino-2-phenylindole (DAPI), with distamycin A (DA) counterstaining, provided the molecular content of some repetitive regions in the karyotypes of the species. One male of E. binotatus presented an extensive heteromorphism, involving at least five different pairs, probably as a consequence of multiple reciprocal translocations. Such rearrangements might be responsible for the multivalent chain seen in the meiosis of this specimen, as well as in another male, although not exhibiting chromosome heteromorphism. The remaining males and those belonging to the other species have always shown 11 bivalents in diplotene and metaphase I cells. In all male specimens, metaphases II presented 11 chromosomes. Despite the observed discrepancies, the five species of Eleutherodactylus have a great uniformity in the 2n=22 karyotypes, suggesting an assemblage of species from southeastern and southern Brazil, in contrast to northern and northeastern assemblage which is characterized by higher diploid numbers. Undoubtedly, B. ternetzi could be included in that proposed assemblage, due to its karyotypic similarity with the Eleutherodactylus species, as evidenced in the present study. This fact strongly supports the close relationships of both genera, previously inferred on the basis of several characters shared by their species.

Behaviour of a full-scale expanded bed reactor with overlaid anaerobic and aerobic zones for domestic wastewater treatment.

This paper presents the behaviour of a full-scale expanded bed reactor (160 m3) with overlaid anaerobic and aerobic zones used for municipal wastewater treatment. The research was carried out in two experimental steps: anaerobic and anaerobic-aerobic conditions, and the experimental results presented in this paper refer to four months of reactor operation. In the anaerobic condition, after inoculation and 60 days of operation, the reactor treating 3.40 kg CODm(-3)d(-1) for thetaH of 2.69 h, reached mean removal efficiencies of 76% for BOD, 72% for COD, and 80% for TSS, when the effluent presented mean values of 225 mg.L(-1) of COD, 98 mg.L(-1) of BOD and 35 mg.L(-1) of TSS. Under these conditions, for nitrogen loading of 0.27 kgN.m(-3)d(-1), the reactor generated an effluent with mean N-org. of 8 mg.L(-1) and N-ammon. of 37 mg.L(-1), demonstrating high potential of ammonification. For the anaerobic-aerobic condition (118th day) the system was operated with thetaH of 5.38 h presented mean removal efficiencies of 84% for BOD, 79% for COD, 76% for TSS, and 30% for TKN. The reactor's operation time was less than two months, which was not long enough to reach nitrification. Regarding the obtained results, this research confirmed that this reactor is configured as a flexible and adequate alternative for the treatment of sewage, requiring relatively small area and only thetaH of 10 h that can be adjusted to the local circumstances.

Development and analysis of anaerobic biofilms onto hydrophobic and hydrophilic surfaces.

Fluorescent in situ hybridization (FISH) with domain and group specific probes that target intracellular 16S rRNA were used to investigate microbial composition of anaerobic biofilms developed on polypropylene (hydrophobic) and glass (hydrophilic) surfaces fitted inside a Modified Robbins Device (MRD). Crushed anaerobic granular sludge was used as inoculum for biofilm development in the MRD. The inoculum and biofilms formed showed nearly the same microbial composition, both were dominated by hydrogenotrophic methanogenic Archaea related to the Methanobacteriaceae as detected by the specific probe (MB1174). This group accounted for 44 to 90% of the DAPI-stained cells. Cells which hybridized to the Bacteria specific probe (EUB338) accounted for 3-18% of the DAPI-stained cells. After the first day of the biofilm formation experiment, a larger number of cells, 4.6 x 10(4) cells mm-2, could be seen colonizing the polypropylene coupon compared to the glass, 8.2 x 10(3) cells mm-2. However, after 9 days these numbers were very similar, i.e. 6.3 x 10(5) cells mm-2 and 7.2 x 10(5) cells mm-2, for the glass and polypropylene coupons, respectively. Our data suggest that the hydrophobicity of the support material did not influence the initial development and the microbial composition of anaerobic biofilms developed in the MRD.

Full scale fluidized bed anaerobic reactor for domestic wastewater treatment: performance, sludge production and biofilm.

This paper describes the performance, sludge production and biofilm characteristics of a full scale fluidized bed anaerobic reactor (32 m3) for domestic wastewater treatment. The reactor was operated with 10.5 m x h(-1) upflow velocity, 3.2 h hydraulic retention time, and recirculation ratio of 0.85 and it presented removal efficiencies of 71+/-8% of COD and 77+/-14% of TSS. During the apparent steady-state period, specific sludge production and sludge age in the reactor were (0.116+/-0.033) kgVSS. kgCOD(-1) and (12+/-5)d, respectively. Biofilm formed in the reactor presented two different patterns: one of them at the beginning of the colonization and the other of mature biofilm. These different colonization patterns are due to bed stratification in the reactor, caused by the difference in local-energy dissipation rates along the reactor's height, and density, shape, etc. of the bioparticles. The biofilm population is formed mainly of syntrophic consortia among sulfate reducing bacteria, methanogenic archaea such as Methanobacterium and Methanosaeta-like cells.

Influence of flocculation conditions in the performance of an experimental domestic sewage treatment plant consisting of an anaerobic expanded bed reactor followed by dissolved air flotation.

This paper presents the results of a study performed with an experimental domestic sewage treatment plant (240 m3 x d(-1) flow) consisting of expanded bed anaerobic reactor (EBAR) followed by dissolved air flotation (DAF) unit. For the flotation step, the anaerobic reactor effluent was previously coagulated with 50 mgFeCl3 x l(-1) and flocculated under different conditions (mean velocity gradient, Gf, and flocculation time, Tf). The Gf values were from 60 to 100 s(-1) associated with 13 and 20 min Tf values. During the tests, the following operational conditions of the flotation unit were maintained: chemical addition (50 mgFeCl3 x l(-1)), 18% recirculation rate associated with a pressure of 450 +/- 10 kPa in the saturation chamber and overflow rate of 180 m3 x m(-2) x d(-1). Temperature ranged from 23.8 degrees C to 30.0 degrees C. Best results were achieved for Gf = 80 s(-1) and Tf = 20 min. For these conditions, the DAF unit removal efficiencies were: 94.4% for chemical oxygen demand (with 53 mg x l(-1) COD residual), 87% for phosphorus (with 0.80 mgP x l(-1) residual), 96.7% for total suspended solids (with 9 mg x l(-1) TSS residual) and 96.4% for turbidity (with 12.9 NTU residual), when the anaerobic reactor effluents have worst quality during the whole day.

Sewage treatment by anaerobic biological process associated with dissolved air flotation.

This paper presents the results of a study performed with a lab-scale batch DAF unit fed with previously coagulated (with FeCl3 or cationic polymer) effluent from a pilot scale up-flow anaerobic sludge blanket (UASB) reactor treating domestic sewage. The adequate coagulation/flocculation conditions--chemical dosage, time (Tf) and mean velocity gradient (Gf) in the flocculation step--and air requirements for flotation process were investigated. Best results were achieved for 65 mg.l-1 of FeCl3 at Tf around 15 min and Gf of 80 s-1. In the assays where only polymer was applied, 7 mg.l-1 of cationic polymer dosage gave optimum removals with Tf around 15 min and Gf of 30 s-1. Air requirements ranged from 9.5 to 19.0 g of air.m-3 wastewater. Best TSS (95% and residual of 2 mg.l-1), COD (85% and residual of 20 mg.l-1) and total phosphate (95% and residual of 0.6 mg.l-1) removals were obtained when applying FeCl3, although the use of cationic polymer also produced good level of TSS (74% and residual of 14 mg.l-1) and COD (75% and residual of 45 mg.l-1) removals. For the UASB-DAF (batch) system and FeCl3, global efficiencies would be 97.2% for COD, 97.9% for phosphate and 98.9% for TSS.

Thoracoscopy talc poudrage : a 15-year experience.

OBJECTIVES: To review our experience with thoracoscopy and talc poudrage during the previous 15 years with regards to efficacy, side effects, morbidity, and mortality. METHODS: Six hundred fourteen consecutive patients (58.6% female; mean age, 54.5 years) underwent thoracoscopy with talc poudrage from August 1983 to May 1999. Of these, 457 patients had malignant pleural effusions, 108 patients had benign pleural effusions, and 49 patients had spontaneous pneumothorax. RESULTS: Sixty-four patients were excluded from evaluation for efficacy: 30 patients (4.9%) because the lung did not expand at the time of the procedure and 34 patients (5.5%) because they died within 30 days of the thoracoscopy. All exclusions were in the malignant group. The overall success rate of the 393 patients with malignant pleural effusions was 93.4%, while the overall success for the 108 patients with benign effusions was 97%, although 7 patients (7%) with benign effusions required a second thoracoscopy. The success rate with pneumothorax was 100%. Major morbidity included empyema in 4%, reexpansion pulmonary edema in 2.2%, and respiratory failure 1.3%. CONCLUSION: Thoracoscopy with talc poudrage is effective in producing a pleurodesis in malignant and benign pleural effusion and in spontaneous pneumothorax. However, it should be noted that the insufflation of talc has a systemic distribution associated with a low rate of morbidity and perhaps does induce ARDS, which is sometimes fatal in a small percentage of patients. Because of these side effects, the search for a better agent should be continued.

Hepatic hydrothorax.

Hepatic hydrothorax (HH) is an uncommon manifestation of cirrhosis with ascites. Pleural effusions form when ascitic fluid moves through diaphragmatic defects that have been opened by increased peritoneal pressure. The diagnosis is established clinically by finding a serous transudate and is confirmed by radionuclide imaging demonstrating communication between the peritoneal and pleural spaces. In end-stage liver disease, the management of hepatic hydrothorax is problematic and often does not respond to medical therapy. Therapeutic options for a refractory hepatic hydrothorax include therapeutic thoracentesis, talc slurry through a chest tube, peritoneovenous and pleurovenous shunting, thoracoscopic talc poudrage, transjugular intrahepatic portosystemic shunt (TIPS), thoracosopic diaphragmatic defect repair followed by talc poudrage, and lastly, liver transplant. TIPS can be used as a bridge for transplantation but is often complicated by encephalopathy. Video assisted thoracic surgery (VATS) with patching the defect and talc poudrage may provide symptomatic relief; however, the morbidity and mortality in these extremely ill patients is high. The only definitive treatment for refractory hepatic hydrothorax associated with end-stage cirrhosis is liver transplantation.